Volume 24, Issue 12, Pages 3115-3124 (September 2018) Stiffness-Induced Endothelial DLC-1 Expression Forces Leukocyte Spreading through Stabilization of the ICAM-1 Adhesome  Lilian Schimmel, Miesje van der Stoel, Carmela Rianna, Anne-Marieke van Stalborch, Aafke de Ligt, Mark Hoogenboezem, Simon Tol, Jos van Rijssel, Robert Szulcek, Harm Jan Bogaard, Patrick Hofmann, Reinier Boon, Manfred Radmacher, Vivian de Waard, Stephan Huveneers, Jaap D. van Buul  Cell Reports  Volume 24, Issue 12, Pages 3115-3124 (September 2018) DOI: 10.1016/j.celrep.2018.08.045 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 24, 3115-3124DOI: (10.1016/j.celrep.2018.08.045) Copyright © 2018 The Authors Terms and Conditions

Figure 1 DLC-1 Expression Is Substrate Stiffness Dependent and Determines PMN Spreading (A) Zooms of left iliac artery obtained from organ donor presented with non-centrical atherosclerotic plaque formation and immunohistochemistry (IHC) stained for DLC-1 and PECAM-1 in the non-plaque region (left panel) and shoulder region (right panel). The open arrowheads indicate EC layer, and the filled arrowheads indicate internal elastic membrane, not visible in the shoulder region due to increased intima thickness. (B) Relative DLC-1 expression in ECs in shoulder and non-plaque regions based on staining intensity and corrected for PECAM-1 intensity; each data point represents one field of view from a total of three patients. (C) Western blot of total cell lysates of lung microvascular ECs from three PAH patients showing increased DLC-1 expression compared to three controls. VE-cadherin staining shows obtained samples from PAH patients are ECs with HeLa as negative control. (D) Quantification of three patients and three controls showing significant increase in DLC-1 expression in PAH patients. (E) Representative western blot of total cell lysates of HUVECs cultured on stiff (plastic) or soft (2 kPa) substrates showing DLC-1 expression. (F) Quantification of three independent experiments showing significant reduced DLC-1 expression on soft substrates. (G) Representative stills from PMN spreading (black-dashed line) and diapedesis (red-dashed line) on HUVECs cultured on stiff (glass) or soft (1.5 kPa) substrates or on stiff (glass) upon DLC-1 depletion with second shRNA (sh2DLC1). (H and I) Quantification shows reduced spreading (H), reflected by PMN surface area and circularity index (I) and spreading on the apical surface of ECs. Data were obtained from three independent experiments with n = 30 cells. (J) Representative force maps recorded with scan size of 30 × 30 μm with 40 × 40 pixels of shCtrl and shDLC-1 ECs showing 1,600 force curves/map. (K) Young moduli of shCtrl or shDLC-1 cells. Values of 12 force maps are plotted as median, and error bars represent 25th and 75th percentile. Data are mean ± SEM. ∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001. Cell Reports 2018 24, 3115-3124DOI: (10.1016/j.celrep.2018.08.045) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Actin Adaptor Protein Recruitment upon ICAM-1 Clustering Is DLC-1 and Substrate Stiffness Dependent (A) Representative western blot of ICAM-1 IP after clustering with αICAM-1 antibody-coated Dynabeads on shCtrl- or shDLC-1-transduced HUVECs. TCL, total cell lysates. (B and C) Quantification of three independent experiments of actin adaptor recruitment upon ICAM-1 IP as indicated (B), and quantification of protein expression levels in TCL (C). (D) Recruitment of filamin A, filamin B, α-actinin-4, and cortactin to ICAM-1 upon clustering with αICAM-1 antibody-coated Dynabeads (IP) on EC cultured on high, medium, or low substrate stiffness. (E and F) Quantification of three independent experiments of actin adaptor recruitment upon ICAM-1 IP (E), and quantification of protein expression levels in TCL (F). (G) Immunoprecipitation with anti-DLC-1 or control IgG (Ctrl) on TNFα-treated HUVECs show interaction between DLC-1 and filamin B and α-actinin-4. Right panels show TCL, and left panel shows IP as indicated. (H) IF images of HUVECs transduced with indicated plasmids showing recruitment of ICAM-1-mCherry and GFP or DLC-1-FL-GFP to αICAM-1 antibody-coated beads after 60 min. (I) Line plot along the white dashed line shows gray values for DLC-1 (green line) above background GFP levels co-localizing with ICAM-1-mCherry (red line) at the edges of the bead (gray-dashed line). (J) Mean gray value within 0.8 μm from gray dashed line in Figure 3I; n = 24 from three independent experiments. Data are mean ± SEM. ∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002. Cell Reports 2018 24, 3115-3124DOI: (10.1016/j.celrep.2018.08.045) Copyright © 2018 The Authors Terms and Conditions

Figure 3 PMN Spreading Is Independent of the GAP Function of DLC-1 (A) TEM flow assay with Ctrl, knockdown, and full-length (FL) or GAP-dead (GD) rescue showing number of adhesive PMNs. (B and C) Same as under (A) but showing PMN numbers that transmigrated (TEM) (B) and showing PMN percentage that finished TEM (C). (D and E) Lateral migration of PMNs on top of ECs from adhesion until diapedesis represented as migration distance in micrometers (D) and migration velocity in micrometers per minute (E). (F) Quantification of percentage of adhesive PMNs, based on spread (open bar) or round (closed bar) phenotype on Ctrl, DLC-1-silenced, FL-rescued, or GD-rescued TNFα-treated ECs. (G–J) Lateral migration tracks of adhesive PMNs before diapedesis on top of Ctrl (G), DLC-1-knockdown ECs (H), DLC-1-FL-rescued ECs (I), or DLC-1-GD-rescued ECs (J); ECs were TNFα-treated. Data were obtained from three independent experiments, two fields of view per experiment for (A)–(F). Data were obtained from three independent experiments, n = 6 tracks condition for (G)–(J). Data are mean ± SEM. ∗∗p < 0.0021, ∗∗∗p < 0.0002. Cell Reports 2018 24, 3115-3124DOI: (10.1016/j.celrep.2018.08.045) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Actin Adaptor Protein Recruitment and PMN Spreading upon Stiffness Substrates Restored upon DLC-1 Overexpression (A) Representative western blot of ICAM-1 IP after clustering with αICAM-1 antibody-coated Dynabeads on Ctrl, knockdown, and FL- or GD-rescued HUVECs showing recruitment of filamin B, α-actinin-4, and cortactin. (B and C) Quantification of three independent experiments of actin adaptor recruitment upon ICAM-1 IP and protein expression levels in TCL for DLC-1-FL rescue (B) and (C) DLC-1-GD rescue (C). (D) Recruitment of filamin A, filamin B, α-actinin-4, and cortactin to ICAM-1 upon clustering with αICAM-1 antibody-coated Dynabeads on Ctrl or DLC-1-GD-GFP-overexpressing ECs cultured on high, medium, or low substrate stiffness, TNFα-treated. (E and F) Quantification of four independent experiments of ICAM-1 IP (E) and TCL (F) shown as fold increase upon DLC-1-GD-GFP overexpression. (G and H) Spreading of PMNs on top of ECs cultured on high, medium, or low substrate stiffness (Ctrl) or on top of ECs overexpressing DLC-1-GD-GFP cultured on high, medium, or low substrate stiffness, shown as PMN surface area (G) or circularity (H); data were obtained from three independent experiments with n = 30 cells per condition. (I) TEM of PMNs on aforementioned conditions (G and H) is quantified as fold increase upon DLC-1-GD-GFP after normalization for high substrate stiffness. Fold increase of >1 indicates increased PMN TEM upon DLC-1-GD-GFP overexpression compared to Ctrl ECs on the same substrate stiffness. Data were obtained from three independent experiments with one field of view per experiment. Data are mean ± SEM. ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001. Cell Reports 2018 24, 3115-3124DOI: (10.1016/j.celrep.2018.08.045) Copyright © 2018 The Authors Terms and Conditions